First Report of Cucumber mosaic virus from Eggplant (Solanum melongena) in Bangladesh

Abstract

In Bangladesh, eggplant (Solanum melongena L.) is largely cultivated by subsistence farmers for domestic consumption and generating family income. During a survey of family-owned farms in April of 2014 in Barisal region of Bangladesh, we observed a farmer's field (0.25 acres) of 3-month-old eggplants with nearly 90% of plants showing mild mosaic and mottling of leaves. Symptomatic plants showed reduced growth, with nearly 50% fewer fruits than from healthy plants. Symptomatic leaves tested positive for Cucumber mosaic virus (CMV; genus Cucumovirus, family: Bromoviridae) by immunostrip diagnostic kit (Agdia, Elkhart, IN). For confirmation of the virus identity, leaf samples were pressed on FTA Plant Cards (Whatman International, Maidstone, UK) and air-dried at room temperature. For eluting total nucleic acids, four to eight disks were punched from the spotted circles of each FTA card using a Harris micropunch (2-mm diameter, Sigma-Aldrich, USA) and soaked for 1 h in 300 μl of extraction buffer (15 mM Na2CO3, 35 mM NaHCO3, 2% [w/v] PVP40, 0.2% [w/v] BSA, 0.05% [v/v] Tween 20, pH 9.6). After vortexing followed by a brief centrifugation, 10 μl of the supernatant was mixed with denaturing buffer (0.1M glycine-NaOH, pH 9.0, 50 mM NaCl, 1 mM EDTA, pH 8.0, 0.5% [v/v] Triton X-100) containing 1% β-mercaptoethanol, incubated at 95°C for 10 min, and kept in ice until use. Denatured sample (2 μl) was subsequently used in reverse-transcription (RT)-PCR using primers CMV-RNA3F (5′-GTAGACATCTGTGACGCGA-3′) and CMV-RNA3R (5′-GCGCGAAACAAGCTTCTTATC-3′) previously reported (2) to amplify a 529-nucleotide (nt) fragment representing the 210-nt intergenic region and the 319-nt partial coat protein (CP) gene of the RNA 3 segment. The amplicons were cloned into pCR2.1 (Invitrogen Corp., Carlsbad, CA), and DNA isolated from four independent clones per amplicon was sequenced in both orientations. The derived sequences (GenBank Accession Nos. KM516898 to KM516901) showed close to 100% identity among themselves and 97% identity with the corresponding sequence of CMV isolate BK16 from cucumber in Thailand (FN552546). These results supported immunostrip diagnostic assays in confirming the presence of CMV in symptomatic samples of eggplants from Barisal. For additional confirmation, a second primer pair (CMV-CP-F: 5′-ATGGACAAATCTGAATCAACCAG-3′ and CMV-CP-R: 5′-TCAAACTGGGAGCACCCCAGAC-3′) was designed using CMV sequences from JN054635 and GU906293 to amplify the full-length CP gene from the same nucleic acid preparations used above. The approximately 657-nt amplicons, representing the full-length CP gene, were cloned, and plasmid DNA from four independent colonies per amplicon wa s sequenced as described above. The derived CP sequences (KM516902 to KM516905) shared 96 and 95% nucleotide and 98.6 and 99.5% amino acid sequence identities with corresponding sequences of CMV isolates from banana (EF178298) and eggplant (GU906293), respectively, from India. Phylogenetic analysis of CP sequences derived from this study with corresponding sequences available in GenBank indicated that CMV from eggplant in Bangladesh aligned closely with CMV subgroup 1B. CMV was previously reported in chili pepper, and tomato from Bangladesh (1) and in eggplant from Israel (4) and India (3). To our knowledge this is the first confirmed report of the occurrence of CMV subgroup 1B in eggplant in Bangladesh. Since no aphids were observed on eggplants, it is likely that CMV was introduced into the farmer's field through seedlings raised from seed carrying the virus. References: (1) A. M. Akanda et al. J. Fac. Agric., Kyushu Univ. 35:151, 1991. (2) C. De Blas et al. J. Phytopathol. 141:323, 1994. (3) S. Kumar et al. Virus Dis. 25:129, 2014. (4) E. Tanne and S. Zimmerman-Gries. Plant Dis. 64:371, 1980.